About 2% of proteins in the human genome are predicted to be modified by prenylation. These include a broad range of substrates, but prominent among them are many key proteins involved in cell signaling, growth regulation, cell progression and differentiation; for example these include the ras proto-oncogene products and the 3 subunits of the heterotrimeric G proteins. The constituent reactions of this processing pathway are the targets of both currently used and investigational anticancer drugs. In addition, one class of target proteins for this modification, the 3 subunits of the heterotrimeric G proteins, are key constituents of the mediators of about half of all clinically useful drugs. Thus the physiological and pathological function of this processing pathway is closely related to the actions of a wide range of therapeutics from those involved in treatment of cardiovascular diseases to those involved in cancer chemotherapy. Prenylation involves multiple enzymatic steps. Following prenylation, the protein is typically proteolytically processed to remove the last 3 amino acids and the new C- terminus is carboxymethylated. We have characterized the heterogeneity of the prenylation of the heterotrimeric G protein 3 subunits and have found biologically significant variation in all three enzymatic reactions of this pathway. To characterize the functional significance of this variation we are concentrating on a specific subset of variably processed proteins (exemplified by G35 in humans) that are processed by prenylation but not by the subsequent reactions in this complex pathway. We are concentrating on this variant because its processing pattern is sequence dependent. About 10% of prenylated proteins have similar sequence determinants. Using G35 as a model protein we will elucidate the functional significance of this variable processing pattern by testing the hypothesis that G35 mediates unique signaling events in cells (Aim 1), by testing the hypothesis that differential proteolysis of prenylated G3 subunits determines their intracellular trafficking and functional site of action (Aim 2) and by identifying and validating protein-protein interactions related to the unique functions of G35 in cells (Aim 3). Finally, we will evaluate the general role of the variant prenyl processing signal in other predicted proteins from the human genome (Aim 4). These studies will extend the known diversity of the prenyl processing reactions and define the functional significance of this variation. The results of this work will have significance for the role of prenyl processing in cells, for how variation of prenyl processing is related to protein function, for the general mechanisms for signaling through heterotrimeric G proteins, for the actions of the large number of therapeutics that exert their effects through heterotrimeric G proteins, and for the mechanism, consequences and utility of anti-cancer drugs that specifically or coincidently target the protein prenylation reactions. PUBLIC HEALTH RELEVANCE: The proposed project studies how a particular kind of lipid molecule referred to as a prenyl group when attached to proteins in cells alters their structure and function. The enzymes that add prenyl groups to proteins and the modified proteins themselves are targets for many different kinds of drugs including those used to treat heart diseases and cancer. The proposed work will increase our understanding of how these drugs work and will help us develop more effective drugs to treat these common illnesses.